Recombinant enzymatically active calpain expressed in a baculovirus system

ABSTRACT

The present invention is directed to mammalian enzymatically active calpain produced in insect cells by recombinant means. Recombinant vectors and baculoviruses containing cDNA encoding the 80 kDa subunit, 30 kDa subunit, and both subunits are described. Methods for producing recombinant enzymatically active mammalian calpain are also described.

This is a continuation, of application Ser. No. 08/454,855, filed May31, 1995, now abandoned, which is a divisional of Ser. No. 08/275,683filed Jul. 15, 1994, all applications hereby incorporated by referencein their entireties.

FIELD OF THE INVENTION

The present invention relates to recombinant enzymatically active humancalpain and its method of preparation using recombinant technology in abaculovirus-insect cell system.

BACKGROUND OF THE INVENTION

A. Calpain

Calpain is a calcium-activated neutral protease, also known as CANP; EC3.4.22.17. It is an intracellular cysteine protease which isubiquitously expressed in mammalian tissues (Aoki et al., FEBS Letters205:313-317, 1986). Calpain has been implicated in many degenerativediseases including, but not limited to, neurodegeneration (Alzheimer'sdisease, Huntington's disease, and Parkinson's disease), amyotrophy,stroke, motor neuron damage, acute central nervous system (CNS) injury,muscular dystrophy, bone resorption, platelet aggregation, andinflammation.

Mammalian calpain, including human calpain, is multimeric. It consistsof two different subunits, which are a 30 kDa subunit and an 80 kDasubunit, and, therefore, is a heterodimer. There are two forms ofcalpain, calpain I (μ-calpain, μCANP) and calpain II (m-calpain, mCANP),which differ in their sensitivities to the concentration of calciumnecessary for activation. Calpain I requires only low micromolarconcentrations of calcium for activation, whereas calpain II requireshigh micromolar or millimolar levels (Aoki et al. supra, and DeLuca etal., Biochim. Biophys. Acta 1216:81-83, 1993). The same 30 kDa subunitis common to both forms. The two human calpains differ in the sequencesof the DNA encoding their 80 kDa subunit, sharing 62% homology. There isevidence that the 80 kDA subunit is inactive, but that it is autolyzedto a 76 kDa active form in the presence of calcium (Zimmerman et al.,Biochem. Biophys. Acta., 1078:192-198, 1991).

R. Siman, in Neurotoxicity of Excitatory Amino Acids, A. Guidotti, ed.,Raven Press , Ltd., New York (1990) reported upon the role of calpain Iin excitatory amino acid (EAA) induced neurotoxicity, eventually leadingto neuronal cell death. Siman advanced the proposition that calpain Iactivation is an early event in the neurodegenerative process and notjust a secondary response to neuronal death. Siman further reported thatonly one highly selective blocker of calpain was available at thattime--calpastatin. However, calpastatin is not readily taken up bycells, as it is a large globular protein of approximately 280 kDa. Simanalso reported that protease inhibitors of broader specificity, includingleupeptin, were unsuccessful in lowering EAA-induced protein breakdownin vivo. Leupeptin was ineffective presumably because it failed to enterthe cells.

Iwamoto et al., Brain Research, 561:177-180 (1991), described thatactivation of calpain may be an important factor in the abnormalproteolysis underlying the accumulation of plaque and tangles in braintissue from people who suffered Alzheimer-type dementia.

Saito et al., Proc. Natl. Acad. Sci. USA, 90:2628-2632 (1993) reportedthat synaptic loss and neuronal cell death correlate strongly with thedegree of cognitive impairment in Alzheimer's disease. They alsoreported that calpain I was significantly activated in human postmortembrain from patients with Alzheimer's disease, and that the degree ofactivation correlated with those regions of the brain showing thegreatest amount of degeneration. It was suggested that the influences ofcalpain activation may contribute to neurofibrillary pathology andabnormal amyloid precursor protein processing prior to causing synapseloss or cell death in the most vulnerable neuronal populations. Becauseof the association between calpain and nerve degeneration diseases,pharmacological modulation of the calpains by inhibitors meritsconsideration as a potential therapeutic strategy in such diseases, forexample, in Alzheimer's disease.

Rami et al., Brain Research, 609:67-70 (1993) reported that both calpaininhibitor I and leupeptin protected neurons against ischemic and hypoxicdamage resulting from ischemia induced by clamping both carotid arteriesand lowering the arterial blood pressure of rats.

Lee et al., Proc. Natl. Acad. Sci. USA, 88:7233-7237 (1991) provideevidence that calcium-activated proteolysis is an important event in theprocess of post-ischemic cell death and they reported that inhibition ofcalcium-activated proteolysis by means of the proteolytic inhibitorleupeptin protected against the degeneration of vulnerable hippocampalneurons after ischemia. Leupeptin was selected because it was the onlyprotease inhibitor that was previously shown to block a trauma-evokedcalpain response in vivo (Seubert et al., Brain Res., 459:226-232,1988). The authors noted, however, that the therapeutic utility ofmodulating calcium-activated proteolysis will probably depend on thedevelopment of more permeable, potent and specific protease inhibitors.

As evident from the foregoing, specific inhibitors of calpain mayprovide a means of treating those neurodegenerative diseases in whichcalpain is implicated. Calpastatin offers limited utility due to itscell impermeability. Protease inhibitors of broader specificities maynot function in vivo and/or may have undesirable side-effects. Thus,other calpain inhibitors must be identified, and a ready, convenient,safe source of calpain will promote the search for such inhibitors.

B. Calpain cDNA

Recombinant enzymatically active human calpain for testing forinhibitors offers the advantages of 1) being a considerably moreconvenient, readily available source of large amounts of enzyme 2) beingeasier to purify and 3) being free from the safety issues which must beaddressed when the source is human tissues, especially human bloodcells, i.e., potentially hazardous viruses. Native human calpain iscurrently isolated from human erythrocytes and can be purified to whatthe authors characterize as apparent homogeneity (Hatanaka et al.,Biomed. Res., 4:381-388, 1983). However, aside from the obvious problemswith source, the purification procedure can be quite tedious, due to thelow levels of calpain relative to the amount of starting material.Furthermore, native calpain is isolated in the presence of an endogenousinhibitor (calpastatin) which must be separated during purification. Agood source of large amounts of enzymatically active calpain wouldgreatly enhance the search for calpain inhibitors by 1) increasing theavailability of calpain for use in reproducible assays for calpaininhibitors and 2) by facilitating crystallization of the enzyme, therebypermitting the design of rational inhibitors. A recombinant system forproduction further facilitates the production of directed mutants toassist in structural studies. Therefore, a recombinant system forproducing active calpain is needed.

The problem in producing enzymatically active calpain by recombinantmeans is that of expressing two different gene products (the 80 kDasubunit and 30 kDa subunit), getting proper processing and folding ofthe individual products, and obtaining the proper combination of the twoproducts to produce enzymatically active molecules. As stated in theprevious discussion, activated calpain has been implicated in thekilling of neuronal cells. Unfortunately, then, any enzymatically activecalpain produced in a recombinant system would be expected to bedeleterious or lethal to that expression system. Any deleterious effectsupon the expression system utilized would be expected to increase asmore of the activated product is expressed. Notably, many mammaliancells produce an endogenous inhibitor of calpain, which may exert animportant control over the activity of an otherwise lethal protease.

Aoki et al., supra, described the complete amino acid sequence of the 80kDA subunit of human calpain I (μCANP) which they deduced from thesequence of a cDNA clone of human calpain. The cDNA clone of humancalpain was isolated from the cDNA library from human skeletal muscleusing a cDNA for the large subunit of rabbit μCANP as a probe.Expression of the cDNA is not reported.

Imajoh et al., Biochemistry, 27:8122-8128 (1988) described the isolationof a cDNA clone for the large subunit of human calpain II from a humanskeletal muscle library probed with chicken CANP and rabbit mCANP. It isreported that the deduced protein had essentially the same structuralfeatures as those described for μCANP and chicken CANP. The amino acidsequence similarities of the human mCANP to human μCANP and chicken CANPwere reported as 62% and 66%, respectively. Expression of the cDNA isnot described or suggested.

Ohno et al., Nucleic Acids Research, 14:5559 (1986) described thesequence of a cDNA coding for the small subunit (30 kDa) of humancalcium activated protease isolated from a human spleen cDNA library.Comparisons with the reported amino acid sequences of rabbit and porcinesequences revealed only 3% differences.

DeLuca et al., supra., reported the molecular cloning and bacterialexpression of cDNA for the rat calpain II (mCANP) 80 kDa subunit. ThecDNA encodes a protein reportedly exhibiting 93% sequence identity withhuman calpain II, and 61% identity with human calpain I. Expression ofthe cDNA was in E. coli bacteria in a phagemid expression vector.Because the expressed product was insoluble and inactive after cellsonication, it could not be used to screen for calpain inhibitors.

C. Baculovirus Expression Systems

V. Luckow, Current Opinion in Biotechnology, 4:546-572 (1993) and Kiddet al, Applied Biochem. and Biotech., 42:137-159 (1993) recentlyreviewed baculovirus systems for the expression of human gene productsand the use of baculoviruses as expression vectors, respectively. Luckowdiscussed the production of a number of different kinds of proteins,including enzymes. However, the production of only one proteolyticenzyme is mentioned, namely, the metalloprotease stromelysin. Unlikecalpain, this enzyme is not multimeric.

Kidd et al., discussed the use of baculovirus-produced proteins forX-ray structural analysis and for assembly of subunits to formfunctional multisubunit molecules. A number of examples displayed theproper assembly of the subunits to produce functional molecules.Although the author broadly stated that baculovirus expression resultsin the structural integrity of the folded molecules and full biologicalfunction in virtually all cases, the assembly of subunits of dimeric ormultimeric enzymes into a functional enzyme was not reported. Further,in other instances involving multisubunit molecules, i.e. Na,K,ATP-ase,the assembly of subunits was sometimes inefficient. (See DeTomaso etal., infra.)

Others have reported the expression of enzymes in the baculovirussystem. Vernet et al., J. Biol. Chem., 27:16661-16666 (1990) describedthe secretion of a papain precursor from insect cells. Papain is acysteine protease. The prepropapain gene was cloned into the transfervector IpDC125 behind the polyhedron promoter. The recombinant constructwas incorporated by homologous recombination into the genome of thepolyhedrosis virus Autographa californica. An enzymatically inactivepapain precursor was recovered from Spodoptera frugiperda Sf9 cellsinfected with the recombinant baculovirus. Proper processing of thepapain precursor to produce an active enzyme did not occur in theinfected cells.

Fertig et al., Cytotechnology, 11:67-75 (1993) described the productionof pro-kallikrein, which is a precursor of kallikrein, a serineprotease. Pro-kallikrein was produced in insect cells from Spodopterafrugiperda (Sf9) and Mamestra brassicae (IZD-Mb503) infected with arecombinant nuclear polyhedrosis virus Autographa californica (AcNPV),strain E2. To obtain an active enzyme, the pro-kallikrein produced wasactivated in vitro using trypsin.

Button et al., Gene, 133:75-81 (1993) described the production of themetalloproteinase GP63 of Leishmania major in a baculovirus-insect cellexpression system. The enzyme was secreted from Spodoptera frugiperda(Sf9) cells infected with a recombinant nuclear polyhedrosis virusAutographa californica (AcNPV) as a latent protease which wassubsequently activated to full proteinase activity by means of HgCl2treatment.

Hirowatari et al., Arch. Virol., 133:349-356 (1993) described theexpression of a polypeptide believed to exhibit two viral proteinaseactivities required for the processing of the viral precursor protein ofhepatitis C virus (HCV). The polypeptide was expressed in the insectcell line Sf21 infected with a recombinant baculovirus. Baculovirustransfer vector pVL941 was utilized. The proteinase activities wereinferred from the presence of a 70 kDa processed protein.

Although the production of enzymatically active multimeric proteases inthe baculovirus system has not, to the inventors' knowledge, beenreported, the baculovirus system has been used to express functional,multimeric enzymes other than proteases. DeTomaso et al., J. Biol.Chem., 268(2):1470-1478 (1993) describe the expression of functional,rat Na,K-ATPase using the baculovirus expression system. An expressionsystem using insect cells was chosen because some insect cells havelittle or no levels of Na,K-ATPase. A baculovirus system was chosensince baculovirus-infected cells produce high levels of foreign protein.Sf-9 cells derived from Spodoptera frugiperda were utilized. Thebaculovirus was Autographa californica. However, because the activity ofenzyme from insect cells was only 20-25% as great as that from dogkidney outer medulla, the authors concluded that a portion of the enzymeexpressed was inactive.

Wen-Ji et al., J. Biol. Chem., 268(13):9675-9680 (1993) describe theexpression of functional mammalian protein farnesyltransferase in abaculovirus system using SF9 cells. The specific activity of theexpressed protein was 510 nM/mg/hr, which is stated to be essentiallyidentical to that reported for the rat brain enzyme. It was noted,however, that the quantities of protein obtained from native tissue didnot previously allow direct assay of the protein concentration, so thisis the first time specific activity of the protein was determined usinga standard protein assay.

There is no disclosure, or suggestion, of expressing an enzymaticallyactive, multimeric, potentially lethal, protease such as calpain in anyexpression system. It was expected that expression of calpain I, inparticular, would be difficult and would require the presence of aninhibitor, because calpain I is activated at extremely low levels ofCa++ that could be achieved during the infection cycle. Surprisingly,the present inventors unexpectedly found that enzymatically activecalpain can be expressed in the baculovirus system, and in the absenceof an inhibitor.

SUMMARY OF THE INVENTION

The present invention is directed to the production of enzymaticallyactive mammalian calpain by recombinant means. The production ofrecombinant enzymatically active human calpain I in abaculovirus-insect-cell system is specifically described. Calpain soproduced can be beneficially used in assays for screening potentialcalpain inhibitors, thus advancing the art by allowing for rapid andefficient selection of calpain inhibitors which can be used to treatthose diseases in which calpain has been implicated, and for providingsufficient calpain to be crystallized for the rational design of calpaininhibitors. Calpain so produced can also be used in other applicationsincluding as a meat tenderizer and a blood clot dissolver.

In one aspect, the present invention is directed to enzymatically activemammalian calpain produced by recombinant technology.

In another aspect, the present invention relates to plasmid vectorscomprising cDNA encoding mammalian calpain for the production ofrecombinant enzymatically active mammalian calpain.

In yet another aspect, the present invention relates to recombinantbaculoviruses comprising cDNA encoding mammalian calpain for theproduction of recombinant enzymatically active mammalian calpain.

In a further aspect, the present invention relates to a method forproducing enzymatically active mammalian calpain by recombinant meansusing a baculovirus-insect cell system.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the cloning strategy for the 30 kDa calpain subunit.

FIG. 2 depicts the cloning strategy for the 80 kDa calpain I subunit.

FIG. 3 depicts the cloning strategy for the double construct.

FIG. 4 depicts the expression of the subunits in Sf21 cells asdetermined by immunoblot.

FIGS. 5 a-d depict measurement of calcium-dependent protease activity.

FIG. 6, a-b, depicts calcium-dependent processing of the inactive 80 kDasubunit to the 76 kDa active form.

FIG. 7 depicts improved expression in serum-free medium.

FIG. 8 depicts measurement of calcium-dependent protease activity of the80 kDA subunit expressed alone.

FIG. 9 depicts the calcium activation profiles of recombinant and nativehuman calpain I.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the production of mammaliancalpain, specifically enzymatically active calpain, by recombinantmeans. "Calpain" as used herein includes both calpain I and calpain IIand refers to the heterodimer consisting of the two subunits. Thesesubunits are the smaller subunit, having a molecular weight ofapproximately 30 kDa, and the larger subunit, having a molecular weightof approximately 80 kDa, depending on its state of activation. Referenceto the 80 kDa subunit includes at least the 77 kDa and 76 kDa formsresulting from autolysis of the 80 kDa subunit. As will be apparent tothose skilled in the art, the subunits from the different mammalianspecies, even the subunits from different tissues of the same mammalianspecies (Hatanaka, supra), can vary in molecular weight. Thesevariations are included.

"Enzymatically active" as used herein refers to the ability tomeasurably hydrolyze at least one known substrate of calpain, includingcalpain itself, i.e., as a result of autolysis. Enzymatic activity canbe measured by any means acceptable to those skilled in the art formaking such determinations including, but not limited to, fluorescentand calorimetric means. That portion of each subunit sufficient tomaintain enzymatic activity as described above is included. The methodaccording to the present invention facilitates the ready determinationof those regions of the coding sequence (cDNA) necessary for enzymeactivity and the changes in activity which can result upon intentionalmutation of the coding sequence. The phrase "enzymatically active uponexpression" as used herein refers to calpain, or a subunit thereof,having measurable enzymatic activity upon expression without requiringany manipulation other than the presence of calcium.

"Expression" as used herein includes, but is not limited to, in vitrotranslation of the cDNA contained in the vectors and viruses accordingto the invention in insect and other cells. Because some Ca²⁺ is usuallypresent during expression, particularly in the instance ofserum-containing medium, the calpain so produced is enzymatically activeupon expression.

The term "recombinant" as used herein includes, but is not limited to, amolecule, microorganism, plasmid, phage, or other vector, containing anew combination of DNA sequences. The term "microorganism" includesviruses and bacteria. The terms "plasmid", "phage", and "vector" areused according to their meanings as known to those skilled in the art asdefined, for example, in A Dictionary of Genetic Engineering, Stephen G.Oliver and John M. Ward, eds., Cambridge University Press, Cambridge,1988 (incorporated herein by reference).

The term "mammalian" includes all animals of the phylogenetic class"mammalian". Preferably, the calpain is recombinant enzymatically activehuman calpain. More preferably, the calpain is recombinant enzymaticallyactive human calpain I.

The baculovirus Autographa californica nuclear polyhedrosis virus(AcNPV), used in the disclosure that follows, is exemplary. However,other baculoviruses such as Bombyx mori nuclear polyhedrosis virus(BmNPV), Heliothis zea nuclear polyhedrosis virus, Lymantria disparnuclear polyhedrosis virus, as well as Orcytes baculoviruses, viruses ofthe Poxviridae and Parvoviradae, Choristoneura, and Amsacta can beconsidered in place of AcNPV. See "Insect Cell Expression Technology",pp 1-40, in Principles and Practice of Protein Engineering, Jeffrey L.Cleland & Charles S. Craik, (Eds.), John Wiley & Sons, 605 Third Avenue,New York, N.Y. 10158-0012.

While cells from the insect Spodoptera frugiperda were used toillustrate the present invention, over 400 insect cell lines have beenestablished and can be used, especially those from Trichoplusia ni. SeeCleland & Craik, supra. Those skilled in the art can readily determinean insect cell suitable for expression. It is also contemplated thatother cells, such as yeast and mammalian cells, can be utilized with theappropriate vector, the selection of which is within the skill in theart.

The heterologous genes to be expressed by the baculoviruses are commonlyunder the control of the polyhedrin or P10 promoters of AcNPV becausethe polyhedrin and P10 genes are not essential for replication ormaturation of the virus and are highly transcribed. This in no waylimits the use of other promoters for the practice of this invention.See Cleland & Craik, supra.

The expression and recovery of recombinant enzymatically active calpainis specifically disclosed. This was unexpected, particularly for calpainI, which is activated in the presence of micromolar amounts of calciumbecause Ca²⁺ is present in the tissue culture medium in which the cellsused for its production are grown. Because infected cells generallybecome "leaky", it was expected that Ca²⁺ would enter the cells from thesurrounding medium in sufficient quantity to activate any calpain Iproduced which, in turn, would be lethal for the cells and would causethe calpain I to digest itself by autolysis.

The recombinantly produced calpain has been determined to be fullyenzymologically active and to have an enzyme activity profile similar tothat of native calpain, which is important for the use of suchrecombinantly derived calpain in the screening of potential therapeuticcalpain inhibitors. The recombinantly produced calpain exhibited similarsensitivity to known calpain inhibitors, and lack of sensitivity toinhibitors of serine or aspartic protease. The amount of calciumrequired to achieve 1/2 V_(max) was essentially the same for both nativeand recombinant calpain. Similarly, the rates of substrate hydrolysiswere similar, as were the specific activities (data not shown). Again,these features are important for full exploitation of the calpainaccording to the invention.

Surprisingly, the specific activity of the 80 kDa 30 subunit alone wasdetermined to be approximately 20-25% that of the heterodimer. Thespecific activity of the 80 kDa subunit dissociated from the nativeheterodimer was previously determined to be only 3% of the heterodimer.(Kikuchi et al. Arch. Biochem. Biophys., 234: 639-645, 1984). Thus, thestructure of the recombinantly produced 80 kDa subunit appears to bedifferent than that of the subunit dissociated from native calpain, andmay more closely represent the structure of the active subunit. Therecombinant production of the calpain subunits, therefore, facilitatesthe study of structure of the individual subunits.

In the present invention, the expression of calpain was achieved by theexpression of both the 80 kDa and 30 kDa subunits in the same insectcells by either co-infecting cells with two separate viruses comprisinga cDNA for each subunit of calpain, or infecting the insect cells with asingle virus comprising cDNA for both subunits. It was also discoveredthat an increased amount of the 80 kDa subunit is expressed when the 30kDa subunit is coexpressed, thus the 30 kDA subunit may have astabilizing effect on the 80 kDA subunit.

When cells are infected with viruses containing both calpain subunits,all infected cells should express both subunits. Regardless of the addedmultiplicity of infection (MOI), when cells are infected with viruscontaining only one subunit or the other, a higher MOI is required toachieve expression of both subunits, e.g., an MOI of 5 for each virus isneeded for 99% of the cells to contain one or more particles of bothviruses and, thus, express both subunits. In a preferred embodiment,expression is effected by coexpression of both subunits in a singlecell.

To construct the recombinant calpain baculoviruses, probes for part ofthe coding regions of both the 30 kDa and the 80 kDa subunits wereprepared by polymerase chain reaction (PCR) from a cDNA library and wereused to screen a human cDNA phage library. Phages containing most ofeach subunit's coding region were isolated and the insert DNA subcloned.Any regions not present in the isolated clones were PCR-amplified fromthe library, sequence-verified, and attached to the partial clones toproduce the entire calpain coding region. The human cDNA library chosenwas a spleen library available from Clontech (Palo Alto, Calif.,#HL1134a). A spleen library was chosen based on the reported abundantexpression of calpain I and II in rat spleen (Murachi, Trends Biochem.Sci. 8:167-169, 1983).

Table I lists the synthetic oligonucleotide primers used for theamplification of portions of the human calpain 80 kDa and 30 kDasubunits. Primers were selected based on the published cDNA sequencesfor the 80 kDa subunit of calpain I (Aoki et al., FEBS Lett.205:313-317, 1986, incorporated herein by reference) and the 30 kDasubunit (Ohno et al., Nucl. Acids Res. 14:5559, 1986, incorporatedherein by reference). Primers for the 80 kDa subunit of calpain I werefurther chosen based on their dissimilarity to the related human calpainII and primers for the calpain II 80 kDA subunit were chosen based ontheir dissimilarity to human calpain I. Internal primers were selectedto be just outside of known restriction endonuclease sites, allowing forsubsequent digestion at those sites for subcloning the PCR fragmentsinto plasmid vectors. All sequences in Table I are reported 5' to 3'. An"S" following the sequence indicates sense. An "AS" indicates antisense.Primers for the 30 kDa subunit are indicated by "30" and primers for the80 kDA subunits of calpain I and II are designated "80I" and "80II",respectively. Parentheticals below the sequence identification numbersrepresent internal laboratory designations and these will be used in theexamples to follow.

                  TABLE I    ______________________________________    Primers Used to Amplify Human Calpain I    ______________________________________    SEQ ID CGGGATCCTT AGGAATACAT AGTCAGCTGC                                     (AS,30)    NO:1   AGCC    (SM-36)    SEQ ID CACCCTGATC TGAAGAC        (S,30)    NO:2    (SM-37)    SEQ ID GTACACTTGA AGCGTGACTT C   (S,80I)    NO:3    (SM-40)    SEQ ID CAGGCAGCAA ACGAAATTGT C   (AS,    NO:4                             80I)    (SM-41)    SEQ ID CGGGATCCTT ATGCAAACAT GGTCAGCTGC                                     (AS,    NO:5   AACC                      80I)    (SM-47)    SEQ ID ATTTGCGGAT GGTCCGGCTC TTGA                                     (AS,    NO:6                             80I)    (SM-49)    SEQ ID CGCGGATCCT ATAAATATGT CGGAGGAGAT                                     (S,80I)    NO:7   CATCACGCCG    (SM-53)    SEQ ID CCGGGATCCT ATAAATATGT TCCTGGTT                                     (S,30)    NO:8    (SM-65)    SEQ ID AACCAGGAAC ATATTTATAG GATC                                     (AS,30)    NO:9    (SM-66)    SEQ ID GGTGGAACGG CCATGCGCAT C   (S,30)    NO:10    (DL-13)    SEQ ID CATTGATGAT GGAGTCAGGA G   (S,80II)    NO:11    (SM-69)    SEQ ID CTGAGAAACA GAGCCAAGAG A   (AS,    NO:12                            80II)    (SM-70)    ______________________________________

All polymerase chain reactions were performed in a thermal cycler(Perkin-Elmer, Norwalk, Conn.) using either 2.5 units of Taq DNApolymerase (Promega, Madison, Wis.), 3 units of UlTma DNA polymerase(Perkin-Elmer, Norwalk, Conn.) or 2 units of Tli DNA polymerase(Promega, Madison, Wis.) in the presence of the supplied buffer, 0.2 mMdNTP's (Taq and Tli DNA polymerases) or 40 μM dNTP's (UlTma DNApolymerase), 0.75-2 mM added MgCl₂, and 0.25 μM of each primer. After aninitial denaturing incubation for 5 minutes at 94° C., 30-35 cycles ofamplifications were performed as indicated below, followed by a finalextension at 72° C. for 7 minutes. The template was 1-10 μl of lambdaphage library, or partially purified phage, added to a minimum of 30 μlof distilled water. DNA was released for amplification by threesubsequent freezings in dry-ice ethanol followed by thawing at 37° C.

EXAMPLE 1 Cloning the Human Calpain 30 kDa Subunit

FIG. 1 depicts the strategy for cloning the human calpain 30 kDasubunit. Primers DL-13 5'>GGTGGAACGGCCATGCGCATC>3' and SM-365'>CGGGATCCTTAGGAATACATAGTCAGCTGCAGCC>3' were used to amplify base pairs#163-805 of the human 30 kDa cDNA from the HL1134a human spleen λgt10library (Clontech Laboratories, Inc., Palo Alto, Calif.) for use as aprobe. Base pair (bp) numbering throughout follows from the assignmentof the initiation codon "ATG" as base pairs #1-3. Conditions for the PCRwere as above using 30 amplification cycles of 1 minute at 94° C., 1minute at 55° C., and 1 minute at 72° C. The 643-bp fragment wasisolated from low-melt agarose after electrophoresis (SeaPlaque-GTG, FMCBioProducts, Rockland, Me.) and purified by extraction withphenol-chloroform. The fragment was then labeled with 32!P-dCTP(Amersham, Arlington Heights, Ill.) by random-primed labeling usingKlenow DNA polymerase following the supplied method (Promega Corp.,Madison, Wis.) and used to screen the library as follows.

The library was plated for screening on the C600hfl host supplied byClontech. Approximately 350,000 plaque-forming units were plated onfourteen, 150 mm diameter petri plates. Duplicate nitrocellulose liftswere prepared from these plates after the procedures described bySambrook et al., Molecular Cloning: A Laboratory Manual, (secondedition) p 1-1626, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Hybridization reactions with the denatured labeled probe werecarried out overnight at 68° C. in 6×SSC, 50 mM sodium phosphate, pH6.8, 10 mg/ml poly A (Sigma Chemical Co., St. Louis, Mo.), 0.2 mg/mlheparin (Sigma Chemical Co., St. Louis, Mo.), and 0.5% SDS, followed bytwo washes with 3×SSC, 0.1% SDS at room temperature and two washes at1×SSC, 0.10% SDS for 30 minutes at 55° C. Labeled plaques were detectedby autoradiography.

Two phages were found to have inserts containing a portion of the cDNAfor the 30 kDa subunit. The entire 5' end of the cDNA, ending with theinternal EcoRI site at bp #488, was present in the lambda phagedesignated 14.2.4. Lambda phage designated 4.1.1 contained most of theprotein coding region. Plaque-purified 4.1.1 phage was then used to PCRamplify the 3' end of the cDNA. Primers SM-37 5'>CACCCTGATCTGAAGAC>3'and SM-36 5'>CGGGATCCTTAGGAATACATAGTCAGCTGCAGCC>3' were used. PrimerSM-36 adds a BamHI restriction site immediately 3' to the stop codon andchanges the stop codon to "TAA". Amplification was carried out withUlTma DNA polymerase (Perkin-Elmer, Norwalk, Conn.) using the suppliedbuffer with the addition of 40 μM dNTP's and 0.75 mM added MgCl₂ (for 10μl of phage template; 1.55 mM final Mg²⁺ concentration). Theamplification cycles were as follows: 2 minutes denaturation step at 97°C. before addition of the polymerase, followed by 30 amplificationcycles of 1 minute at 95° C., 45 seconds at 55° C. and 1 minute at 72°C. The fragment obtained was then digested with EcoRI and BamHI,isolated from low-melt agarose as above and subcloned into EcoRI,BamHI-digested pGEM-4Z (Promega Corp., Madison, Wis.).

The 5' portion of the gene was obtained in two steps. First, DNAisolated from plaque-purified lambda phage 14.2.4 was digested withHindIII, which cuts the lambda DNA in the region 5' to the insert, andBglII, which cuts the lambda DNA in the region 3' to the insert. Theresulting 2.5 kb fragment was subcloned into BamHI, HindIII-digestedpGEM-4Z. A pair of synthetic oligonucleotides were then used to modifythe region 5' to the start codon by both adding a BamHI site tofacilitate cloning into the transfer vectors and inserting the sequenceCCTATAAAT from the polyhedrin gene 5' untranslated region immediatelybefore the start codon in an attempt to achieve optimal baculovirustranslation. The plasmid containing the lambda insert was digested withXmaI, which cuts in the multiple cloning site of the pGEM-4Z vector 5'to the BamHI site, and HpaI, which cuts at base pair #13 in the 30 kDacDNA, which is 3' to the BamHI site, in order to remove the calpain cDNAsequences 5' to the HpaI site. The digested plasmid was isolated fromlow-melt agarose as above. Two oligonucleotides, SM-655'>CCGGGATCCTATAAATATGTTCCTGGTT>3' and SM-665'>AACCAGGAACATATTTATAGGATC>3' were annealed to one another as shown inFIG. 1 by co-incubation in 10 mM Tris-HCl, pH 7.0, 50 mM NaCl at thefollowing temperatures for 10 minutes each: 90° C., 65° C., 42° C., 37°C., and room temperature. The annealed oligonucleotides were thenligated to the linearized, digested plasmid and the resulting colonieswere screened for the presence of the BamHI site that is added by theseoligonucleotides (FIG. 1).

The cDNA of the entire coding region was then assembled from the twoplasmids described above. The plasmid containing the 5' portion of thecDNA was digested with EcoRI. There is an EcoRI site in the vectormultiple cloning region 5' to the XmaI site and also a site at bp #488in the 30 kDa cDNA. This approximately 500 bp EcoRI fragment containingall the additions to the 5' end of the 30 kDa cDNA was then isolated 20from low-melt agarose as above and ligated to the EcoRI-digested plasmidcontaining the 3' portion of the cDNA. A plasmid with the EcoRI fragmentin the correct orientation was obtained. Dideoxynucleotide DNAsequencing (Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467,1977) of the entire 30 kDa coding region verified that the modified cDNAencoded the correct amino acid sequence for the human calpain protein(Ohno et al., Nucl. Acids Res. 14:5559, 1986).

This plasmid was then digested with BamHI and the 820-base-pair fragmentcontaining the entire human 30 kDa calpain cDNA with its modificationsfor baculovirus expression: 1) addition of CCTATAAAT immediately 5' tothe start codon to potentially improve transcription; and 2) changingthe stop codon to the baculovirus-preferred TAA (Luckow et al.,Virology, 170:31-39, 1989) was subcloned for single subunit expressioninto BamHI-digested pVL941 transfer vector containing the polyhedrinpromoter. The resultant plasmid was designated pVL941-hCANP30-6. Fortwo-subunit expression, the 820-bp fragment was subcloned into eitherBamHI- or BglII-digested pAcUW51 (PharMingen, San Diego, Calif.;designated p51-Bam-CANP30 and p51-Bgl-CANP30, respectively), a transfervector containing both the p10 and polyhedrin promoters (see FIG. 3).The pAcUAW-51 vector is designed to express two proteins simultaneouslyfrom the same virus, inserting both into the polyhedrin locus ofbaculovirus. This vector contains two promoters--polyhedrin andp10--which are strong promoters that begin transcription very late ininfection (i.e., after 18-24 hours). The promoters are inserted in thevector in opposite orientation to minimize deletion of the cDNAs byhomologous recombination due to duplication of the genetic material(Weyer et al., J. Gen. Virol., 70: 203-208 (1991). Resulting plasmidswere verified as having only one insert in the correct orientation forexpression by restriction enzyme analysis (data not shown).

EXAMPLE 2 Cloning the Human Calpain 80 kDa Subunit

FIG. 2 depicts the strategy for cloning the human 80 kDa calpain Isubunit. Primers SM-40 5'>GTACACTTGAAGCGTGACTTC>3' and SM-415'>CAGGCAGCAAACGAAATTGTC>3' were prepared and used to amplify base pairs#1372-2037 of the human 80 kDa cDNA from the HL1134a human spleen λgt10library (Clontech Laboratories, Inc., Palo Alto, Calif.) for use as aprobe. Conditions for the PCR were as above for Taq DNA polymerase withthe solution being made 2 mM with respect to MgSO₄ and the addition of 5μl of lambda library using 30 amplification cycles of 1 minute at 94°C., 1 minute at 60° C. and 2 minutes at 72° C. The PCR-amplifiedfragment was isolated away from primers following the suppliedinstructions with the Wizard PCR prep kit (Promega, Madison, Wis.). Thisfragment was digested with XmaI and SalI. The resulting 538 bp fragmentwas isolated from a 1% Seakem-GTG/2% NuSieve (FMC BioProducts, Rockland,Me.) agarose gel using the supplied protocol for the GeneClean II kit(B101, La Jolla, Calif.) and then ligated to XmaI-, SalI-digestedpGEM-4Z vector (Promega, Madison, Wis.).

DNA from the above partial cDNA clone of the 80 kDa subunit was digestedwith EcoRI and HindIII to release the insert, followed by isolation ofthe fragment using the GeneClean II kit after electrophoresis in a 1%Seakem-GTG/2% NuSieve agarose gel. It was then labeled with 32!P-dCTPand used to screen the human spleen library as described above.Approximately 500,000 plaque forming units were plated on a ten, 150 mmdiameter, petri plates. Duplicate nitrocellulose lifts were prepared andhybridized with the denatured labeled probe overnight at 68° C. in thesame hybridization mix as described above for the 30 kDa screen,followed by two washes with 2×SSC, 0.1% SDS for 15 minutes each at roomtemperature and two washes with 1×SSC, 0.1% SDS for 1 hr each at 68° C.,with detection of the labeled plaques by autoradiography.

One phage (lambda cal80-8a) was found to have an insert containing 67%of the 3' end of the coding region of the cDNA for the 80 kDa subunit.DNA was isolated from a liquid culture of plaque-purified phagefollowing methods described in Sambrook et al., supra, digested withXbaI and SalI, and the unique 1238 bp 80 kDa cDNA fragment was isolatedfrom a 1% SeaKem-GTG agarose gel using the supplied protocol for theGeneClean II kit (B101, La Jolla, Calif.). This fragment was ligated toXbaI-, SalI-digested pBluescript® SK+vector (Stratagene, La Jolla,Calif.) and the identity of the insert verified by dideoxynucleotide DNAsequencing (Sanger et al., supra) of portions of the insert.

The other sections of the coding region of the cDNA were generated byPCR amplification either from the human spleen library (5' end) or theplaque-purified lambda phage cal80-8a (3' end). Amplification of the 3'end was done to remove 3' untranslated sequences and change the stopcodon to the baculovirus-preferred TAA. For the former, primers SM-535'>CGCGGATCCTATAAATATGTCGGAGGAGATCATCACGCCG>3' and SM-495'>ATTTGCGGATGGTCCGGCTCTTGA>3' were used to amplify base pairs #1-1096of the human 80 kDa cDNA from 10 ml of the HL1134a human spleen library.Primer SM-53 adds a BamHI site to facilitate subsequent cloning andinserts the sequence CCTATAAAT immediately before the start codon in anattempt to achieve optimal baculovirus translation. For the latter,primers SM-40 5'>GTACACTTGAAGCGTGACTTC>3' and SM-475'>CGGGATCCTTATGCAAACATGGTCAGCTGCAACC>3' were used to amplify base pairs#1372-2143 of the human 80 kDa cDNA from 1 μl of lambda phage cal80-8a.Amplifications were carried out with UlTma DNA polymerase (Perkin-Elmer,Norwalk, Conn.) using the supplied buffer with the addition of 40 μMdNTP's and 1.5 mM added MgCl₂. The amplification cycles were as follows:5 minutes denaturation step at 95° C. before addition of the polymerase,followed by 30 amplification cycles of 1 minute at 94° C., 1 minute at60° C., and 2 minutes at 72° C.

The 1096 bp fragment with the 5' end was isolated using the GeneClean IIkit after electrophoresis in a 1% Seakem-GTG/2% NuSieve agarose gel.This fragment was then digested with BamHI and XbaI, repurified afterdigestion using the GeneClean II kit and subcloned into BamHI-,XbaI-digested pBluescript® SK+vector (Stratagene, La Jolla, Calif.). The3' end PCR-amplified fragment was isolated away from primers followingthe supplied instructions with the Wizard PCR prep kit (Promega,Madison, Wis.). This fragment was then digested with SalI and BamHI, theresultant 137 bp fragment isolated from a 1% Seakem-GTG/2% NuSieve (FMCBioProducts, Rockland, Me.) agarose gel using the supplied protocol forthe Mermaid kit (B101, La Jolla, Calif.) and then ligated to SalI-,BamHI-digested pBluescript® SK+vector (Stratagene, La Jolla, Calif.).Dideoxynucleotide DNA sequencing (Sanger et al., supra) of both of theseinserts verified that they encoded the correct amino acid sequence (Aokiet al., FEBS Lett. 205: 313-317, 1986) for that portion of the proteinand was used to eliminate clones with mutations from the polymerasechain reaction amplification.

The BamHI, XbaI fragment with the modified 5' end of the coding region,the XbaI, SalI fragment with the middle of the coding region, and theSalI, BamHI fragment from the 3' end of the coding region were digestedwith the appropriate enzymes from their vectors and the fragmentsisolated from a 1% Seakem-GTG/2% NuSieve (FMC BioProducts, Rockland,Me.) agarose gel using the supplied protocol for the GeneClean II kit(B101, La Jolla, Calif.). These fragments were then mixed in equimolaramounts with pVL941 vector (Luckow and Summers, Virology 170:31-39,1989) that had been digested with BamHI and treated with shrimp alkalinephosphatase (U.S. Biochemical Corp., Cleveland, Ohio) following themanufacturer's protocol and ligated together. A clone (plasmiddesignation pVL941-hCANPI80-4) containing the correctly-sized BamHIfragment in the proper orientation, as determined by restriction enzymeanalysis, was used for the production of the recombinant baculovirusexpressing only the 80 kDa subunit (see below).

Plasmid PVL941-hCANI80-4 was also digested with BamHI and the 2153 bpfragment containing the entire human 80 kDa calpain I cDNA was subclonedinto either BamHI-digested p51-Bgl-CANP30 or BglII-digestedp51-Bam-CANP30 for the production of single vectors containing cDNAs forboth subunits, i.e. double constructs (see FIG. 3). The resultingplasmids (designated p51-hCANPI-1 and p51-hCANPI-2, respectively) wereverified as having only one new insert in the correct orientation forexpression by restriction enzyme analysis.

The above represents the method that was used to clone the cDNAs forhuman calpain I. There are a number of comparable methods known to thoseskilled in the art that could allow one to obtain cDNA sequences ofthese genes suitable for recombinant expression. Similar methods couldalso be used to obtain the cDNA for calpain II for recombinantexpression.

The same strategy was used to generate a probe for library screening toobtain the human calpain II 80 kDa cDNA. The cDNA for the coding regionof the 80 kDa subunit of calpain II is reported in Imajoh et al., supra(incorporated herein by reference). Primers SM-695'>CATTGATGATGGAGTCAGGAG>3' and SM-70 5'>CTGAGAAACAGAGCCAAGAGA>3' wereused to amplify bp #1587-2075 from the same human spleen library.Conditions for the PCR are described above using 2 units of Tli DNApolymerase (Promega, Madison, Wis.) with 0.75 Mm added MgCl₂ and 10 μlof lambda library using 30 amplification cycles of 1 minute at 94° C., 1minute at 55° C. and 1 minute at 72° C. The 489 bp fragment was isolatedfrom a 1% Seakem-GTG/2% NuSieve (FMC BioProducts, Rockland, Me.) agarosegel using the supplied protocol for the GeneClean II kit (Bio101,LaJolla, Calif.), digested with PstI and BamHI, and the procedure justdescribed used to isolate the 377-bp PstI-, BazEI-fragment after agarosegel electrophoresis. The purified fragment was ligated to PstI-,BamHI-digested pGEM-4Z vector (Promega, Madison, Wis.).

EXAMPLE 3 Production of Recombinant Baculoviruses

Spodoptera frugiperda cells (Sf21; Vaughn et al., In Vitro, 13:213-217,1977) were provided by Dr. B. G. Corsaro of the Boyce Thompson Institutefor Plant Research, Cornell University, Ithaca, N.Y. These cells weregrown in suspension at 27° C. in supplemented Grace's medium (JRHBiosciences, Lenexa, Kans.) with the addition of 10% defined fetalbovine serum (Hyclone Laboratories, Inc., Logan, Utah). Monolayercultures for some expression studies and plaque assays were obtained byseeding the suspension-grown cells in tissue culture flasks at thedensities indicated for the applications.

Recombinant baculoviruses were produced by cotransfecting Sf21 cells ina monolayer culture (approximately 2×10⁶ cells in a 25 cm² flask) with0.5 mg of linearized AcNPV DNA (Baculogold®, PharMingen, San Diego,Calif.) and 2 mg of one of the four vectors described above (listedbelow in Table II) using Insectin® liposomes following the suppliedprotocol from InVitrogen (San Diego, Calif.). The resulting culturesupernatant containing primarily recombinant baculoviruses was harvested2-5 days later and used to set up plaque plates of the extracellularvirus.

                  TABLE II    ______________________________________    Vector          Calpain subunit                               Promoter    ______________________________________    pVL941-hCANP30-6                    30 kDa     polyhedron    pVL941-hCANPI80-4                    80 kDa     polyhedron    p51-hCANPI-1    30 kDa     polyhedron                    80 kDa     p10    p51-hCANPI-2    30 kDa     p10                    80 kDa     polyhedron    ______________________________________

Sf21 cells were seeded in 60-mm culture dishes (2×10⁶ cells/dish) andinfected for one hour with 1 ml of 10-fold serial dilutions of thecotransfection culture supernatant (10⁻² to 10⁻⁵) and subsequentlyoverlaid with 4 ml of a 1:1 mixture of 2× supplemented Grace's medium(Gibco BRL, Gaithersburg, Md.) and 2% Seakem agarose (FMC Bioproducts,Rockland, Me.). Putative recombinant plaques were identified 5-7 dayslater by visual inspection for occlusion-body-negative plaques usingboth a dissection and an inverted-phase microscope following 7 minutesstaining with 0.05% neutral red in Dulbecco's PBS. Plaques were verifiedas being recombinant by the hybridization of 32!P-labeled 30 kDa or 80kDa human calpain I sequences to blots of infected cell lysates (Summersand Smith, A Manual of Methods for Baculovirus Vectors and Insect CellCulture Procedures, Texas Agricultural Experiment Station Bulletin 1555:1-57, 1987). Recombinant virus was then expanded for the first twopassages in monolayer Sf21 cultures, with subsequent virus passages(minimum of three to a maximum of five) using suspension Sf21 cultures.All virus expansions were carried out by infecting Sf21 cells at amultiplicity of infection (MOI) of less than 0.5 and collecting themedium containing the extracellular virus particles 3-4 days afterinfection.

Seven independent plaque-pure recombinant viruses (recombinant virusesdesignated AcNPV-hCANP30-1 through -7, respectively) were isolated fromthe transfection of cells with pVL-hCANP30-6. AcNPV-hCANP30-5 wasdeposited Jun. 2, 1994, with the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852-1776 (hereinafter "ATCC") and bearsATCC designation ATCC VR 2459. Six independent plaque-pure recombinantviruses (recombinant viruses designated AcNPV-hCANPI80-1 through -6,respectively) were isolated from the transfection of cells withpVL-hCANPI80-4, all of which contained the DNA for the 80 kDA subunitonly. AcNPV-hCANPI80-5 was deposited on Jun. 2, 1994, with the ATCC andbears ATCC designation ATCC VR 2457. Twelve independent plaque-purerecombinant viruses (designated AcNPV-hCANPI-1-2, 1-5, 1-7 and 1-8 andAcNPV-hCANPI-2-1 through 2-8, respectively), were isolated from thecotransfections of cells with p51-hCANPI-1 and p51-hCANPI-2. Of the 12recombinant viruses isolated, only 5 contained the DNA for both the 30kDa and 80 kDa subunits. These 5 recombinant viruses wereAcNPV-hCANPI-1-5, ACNPV-hCANPI-1-7, AcNPV-hCANPI-1-8, AcNPV-hCANPI-2-3,and AcNPV-hCANPI-2-5. AcNPV-hCANPI-2-5 was deposited on Jun. 2, 1994,with the ATCC and bears ATCC designation ATCC VR 2458. The eighteenselected recombinant viruses obtained were then examined for theirability to express the calpain protein as described below. All depositswere made under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure, all aspects of which are hereinincorporated by reference. All deposits were tested by the ATCC anddetermined to be viable at the time of deposit.

EXAMPLE 4 Expression and Recovery of Baculovirus Recombinant Calpain

Sf21 cells were seeded in 24 well plates at 1.5×10⁵ cells/cm² insupplemented Grace's medium with 10% fetal bovine serum. After cellattachment, virus (see below for specific virus designations) was addedat an MOI of between 1 and 5. The cells were harvested sometime after 24hours. With the present expression system, optimal yield was achievedwith harvests between 36-48 hours after infection. For the harvest, alysis buffer of 50 mM Tris-HCl, 10 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF), 1 μg/ml leupeptin, and 0.1% NP-40, pH 7.4 was used,followed by centrifuging the homogenates in an Eppendorf tube at14,000×g for 10 minutes at 4° C. and recovering calpain and othercellular proteins. Proteins were denatured by adding 0.2% SDS andheating the samples for 5 minutes at 95° C. Samples were then stored at-70° C. prior to analysis.

The calpain expressed and recovered was examined by immunoblot analysisas follows. Ten to twenty micrograms of total protein was separated bySDS-PAGE (Laemmli, 1970) using 10% or 12.5% acrylamide gels andtransferred to 0.45 mm nitrocellulose (Bio-Rad, Melville, N.Y.) by themethod of Towbin et al., Proc. Natl. Acad. Sci. USA 76: 4350-4354(1979). Calpain protein was specifically detected using a 1:1,000dilution of polyclonal anti-calpain serum which detects both subunits(Siman et al., J. Neurosci. 10:2400-2411, 1990). The antiserum wasdiluted in 20 mM Tris-HCl, pH 7.4, with 150 mM NaCl and 5% Carnationnonfat dry milk (blocking buffer). Non-specific antibody binding wasremoved by washing with 20 mM Tris-HCl, pH 7.4, with 150 mM NaCl and0.05% Tween-20. Alkaline-phosphatase-conjugated goat anti-rabbit IgG(Bio-Rad, Melville, N.Y.), diluted 1:2,000 in blocking buffer, was thenadded. The secondary antibody was detected using the alkalinephosphatase conjugate substrate kit (Bio-Rad, Melville, N.Y.). Theresults for recombinant viruses AcNPV-hCANP30-5, AcNPV-hCANPI80-5, andAcNPV-hCANPI-2-5 are shown in FIG. 4.

The expression of the appropriate individual calpain subunit resultingfrom cells infected with the AcNPV-hCANP30-5 and AcNPV-hCANPI80-5viruses alone are depicted in lanes 2-3 and 4-5, respectively. When thecells were coinfected with two viruses, one containing the DNA constructfor the 30 kDa calpain subunit (AcNPV-hCANP30-5) and the othercontaining the DNA construct for the 80 kDa calpain subunit(ACNPV-hCANPI80-5), both appropriate calpain subunits were expressed.This is depicted in lanes 6-8. Similarly, when cells were infected withAcNPV-hCANPI-2-5, which contained the DNA construct for both the 30 kDaand 80 kDa calpain subunits, both ubunits were expressed (lanes 9-10).The ability of an anti-calpain serum to detect the recombinant proteinverified that authentic calpain protein was produced. Lane 1 representsinfection with wild-type virus. No calpain expression was detected.

Surprisingly, the accumulated amount of the 80 kDa ("catalytic") subunitwas unexpectedly increased (as determined by visual inspection) bycoexpression with the 30 kDa ("regulatory") subunit, either bycoinfection of cells with AcNPV-hCANP30-5 and AcNPV-hCANP80-5 (lanes6-8) or by expression of the double construct AcNPV-hCANPI-2-5 (lanes9-10), as compared with expression in the absence of the 30 kDa subunit(lanes 4-5). Previous research into the role of the 30 kDa subunit wouldnot allow one to predict this stabilizing effect on the other subunit.

Analysis of all of the AcNPV-hCANP30 and the AcNPV-hCANP80 recombinantviruses in the same fashion as above showed comparable levels ofexpression of their respective subunits (data not shown). While all fivevirus isolates of AcNPV-hCANPI containing both calpain subunitsexpressed the intact 80 kDa calpain subunit, four of the isolatedviruses (i.e., AcNPV-hCANPI-1-5, AcNPV-hCANPI-1-7, AcNPV-hCANPI-1-8, andAcNPV-hCANPI-2-3) failed to also express detectable amounts of the 30kDa calpain subunit (data not shown). The absence of the 30 kDa subunitwas further evident by the decreased level of expression of the 80 kDasubunit, which was comparable to that seen with the AcNPV-hCANPI80viruses, as opposed to the increased level observed with coinfections ofAcNPV-hCANP30 and AcNPV-hCANPI80 and with infection withAcNPV-hCANPI-2-5. That only 5 out of 12 of the double subunit virusesstill contained DNA sequences for both subunits following recombinationand selection of recombinant viruses and that, of those 5, only one wasfound to coexpress both protein subunits, suggests the existence of aselection pressure against the insect cell expression of the completetwo-subunit calpain protease. This might be a consequence of thelethality of calpain.

The additional bands visible around the 80 kDa subunit bands in FIG. 4are a result of the autolytic enzyme activity. The two bands visible inlanes 6-8 represent the 80 kDa and autolytically-resultant 76 kDa forms,respectively. Two bands are also observed on the blot for lanes 9 and 10but may not be visible in the Figure. Three bands are actually visiblein lanes 4 and 5. In addition to the 80 kDa and autolytically-resultant76 kDA forms, a stable intermediate of 77 kDa forms at low enzymeconcentration and was previously reported to form upon autolysis of thelarge subunit in a calpain I heterodimer incubated with calcium underdilute conditions. (Inomata, et al., J. Biol. Chem., 263:19783-19787,1988.) As is apparent from FIG. 4, however, most of the recombinanthuman calpain recovered is in the 80 kDa form, which is desired in theinstance of calpain considering the potential lethality to the system.Since calpain autolyses to the active form in the presence of calcium,no separate treatment is required for activation other than addingcalcium.

EXAMPLE 5 Enzyme Activity of the Baculovirus Recombinant Calpain

Sf21 cells were again seeded in 24 well plates at 1.5×10⁵ cells/cm² andinfected with either wild-type virus, coinfected with bothAcNPV-hCANP30-5 and AcNPV-hCANPI80-5 (MOI of 5 for each virus), orinfected with AcNPV-hCANPI-2-5 (MOI=5.7). The intracellular proteinswere harvested at 40 hours after infection with 100 μl/well of thefollowing lysis buffer: 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1mM EGTA, 5 mM β-mercaptoethanol, 0.1% Triton-100, followed by a 10minutes centrifugation at 14,000×g at 4° C. to pellet the nuclei andsome membranes. The in vitro enzyme activity of 20 μl of each extract(12.5-20 μg of total protein) was measured using the synthetic peptidesubstrate succinyl-leucine-tyrosine-aminomethyl coumarin(Succ-Leu-Tyr-AMC) at a 1 mM concentration with and without the additionof 5 mM CaCl₂ following the procedure of Sasaki et al., J. Biol. Chem.,259:12489-12494 (1984), incorporated herein by reference. The activityobtained was compared with the enzyme activity of 2 μg ofpartially-purified native human calpain I produced according to themethod of Siman et al., supra.

The activity in the presence of calcium was also measured with theaddition of 12.5 μM calpain inhibitor I following the proceduredisclosed in Sasaki et al., supra. The results are depicted in FIGS.5a-d. In FIGS. 5a-d, open circles represent activity without calciumpresent. Solid diamonds represent activity with calcium present.Crossed-squares represent activity with calcium and calpain inhibitor Ipresent. The data in FIGS. 5a-d show 1) a calcium-dependent increase insubstrate hydrolysis by native human calpain I that is inhibited bycalpain inhibitor I (5a); 2) no endogenous calcium-dependent substratehydrolysis in cells infected with wild-type virus (5b); and 3) in cellseither coinfected with AcNPV-hCANP30 and AcNPV-hCANP80 (5c) or infectedwith AcNPV-hCANPI-2-5 alone (5d), there is a calcium-dependent increasein substrate hydrolysis that is also inhibited by calpain inhibitor I,just as seen with the native enzyme. Based on these results, therecombinantly produced calpain has the same enzyme activity profile asnative calpain. Again, this is important for effective utilization ofthe recombinant enzymatically active calpain to screen potential calpaininhibitor therapeutics.

EXAMPLE 6 Autolytic Enzyme Activity of the Baculovirus RecombinantCalpain

The autolytic enzyme activity of the recombinant calpain wasdemonstrated by showing that the recombinant calpain correctlyautoprocessed the 80 kDa subunit to the "activated" 76 kDa form. The 76kDa form is produced in the presence of calcium by autolytic cleavageand is indicative of active enzyme. Lysates from cells either infectedwith wild-type AcNPV, coinfected with both AcNPV-hCANP30-5 andAcNPV-hCANPI80-5 or infected with AcNPV-hCANPI-2-5 prepared in Example 5were incubated with or without the addition of 6.7 mM

CaCl₂ for 5 minutes at room temperature, stopping the reaction by adding6.7 mM EDTA and boiling the samples in SDS-PAGE-gel-loading buffer priorto storage at -70° C. The results of PAGE of the preparations aredepicted in FIGS. 6a and b. In FIGS. 6a and b, the presence and absenceof calcium is indicated by a "+" or "-", respectively. FIG. 6a depictsan anti-calpain I immunoblot with and without in vitro calciumincubation. The AB#4 antiserum preparation used was raised againstpurified native calpain. It primarily detects the 80 kDa subunit,although it will also bind the 76 kDa autolytic cleavage product. Lanes1 and 2 contain the partially purified native human calpain I 80 kDasubunit (purified from human red blood cells as described previously,see Kitahara, et al., supra). Lane 3 contains the protein fraction fromcells infected with the wild-type virus. Lanes 4 and 5 represent theprotein fraction from cells cotransfected with AcNPV-hCANP30-5 andAcNV-hCANPI80-5. Lanes 6-7 represent the protein fraction from cellstransfected with AcNPV-hCANPI-2-5. In the absence of added calcium,lanes 4 and 6 have two immunoreactive bands. Lane 1 (the native humancalpain I purified from human red blood cells) has only one band. Theupper bands of lanes 4 and 6 comigrate with the 80 kDa subunit of thenative partially-purified human erythrocyte calpain I and the lowerbands comigrate with the 76 kDa native human calpain I that has beenincubated with calcium (lane 2). The lower bands, present even in theabsence of calcium, appear to be endogenously activated calpain,presumably due to the intracellular influx of calcium from the medium assome cells become leaky during the infection. A single band is detectedin all the samples incubated with calcium (lanes 2,5, and 7), and itcomigrates with the 76 kDa native human calpain I (lane 2) that has alsobeen incubated with calcium.

The data in FIG. 6b show that the protein migrating as a 76 kDa band isthe properly-cleaved authentic 76 kDa autocatalytic fragment. FIG. 6bcontains the results of an immunoblot analysis of the same samples as in6a except that the AB#34 antibody used was generated against the firstfive amino acids at the N-terminus of the 76 kDa fragment (anti-LGRHEC);(Saido et al., J. Biochem. 111:81-96 (1992)). This antibody specificallyrecognizes only the properly-cleaved native human enzyme (i.e., 76 kDA;lane 2) and not the intact 80 kDa calpain I (lane 1). Both the smallamounts of the endogenously-cleaved recombinant calpain I large subunitand the single abundant 76 kDa band after the addition of calcium aredetected by this antiserum. The foregoing results demonstrate that theentire amount of the 80 kDa subunit recombinant protein is capable ofbeing autocatalytically activated by the addition of calcium and thatthe activated subunit is properly cleaved.

EXAMPLE 7 Improved Expression in Serum-Free Medium

Spinner cultures of insect cells are routinely used for the baculovirusexpression of recombinant proteins because of the greater ease inhandling large numbers of cells as compared to monolayer cultures. Acomparison was made between the production of calpain in Sf21 cellsgrown in supplemented Grace's medium with 10% defined fetal bovine serumversus Sf21 cells adapted to serum-free medium by serial two-folddilutions to growth in ExCell-401 (JRH Biosciences, Lenexa, Kans.).Log-phase Sf21 cells grown in either medium were centrifuged at 150×gfor 10 minutes to pellet the cells and resuspended at 10⁷ cells per mlin their growth medium containing AcNPV-hCANPI-2-5 virus at an MOI=2.Cells plus virus were incubated at room temperature for 1 hour with anoccasional gentle resuspension of the cells by hand. The entire mixturewas added to the appropriate medium in 250 ml spinner flasks (Techne,Inc., Princeton, N.J.) to achieve a final volume of 100 ml at a celldensity of 1.5×10⁶ cells per ml. Duplicate infections for each mediumwere incubated at 27° C. with stirring, at a speed of 80 rpm for theserum-containing cultures and at 100 rpm for the serum-free cultures.Cultures were sampled at 24 hours and 48 hours.

Samples were harvested to permit measurement of the enzyme activity asdescribed in Example 5, following pelleting of the cells by centrifugingat 150×g for 10 minutes, resuspension in Dulbecco's phosphate-bufferedsaline (Mediatech, Inc., Herndon, Va.), and then again centrifuging at150×g for 10 minutes. Total protein concentrations were measured usingthe Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Melville N.Y.)following the supplied protocol, with bovine serum albumin as thereference protein standard. The serum-free-adapted cells hadunexpectedly low levels of recombinant calpain protein and activity at24 hours, but unexpectedly higher levels than the serum cultures at 48hours (FIG. 7). More importantly, there was proportionately less of theactivated 76 kDa protein at 48 hours in the serum-free cultures ascompared to the serum-containing cultures (data not shown). The higheramount of activated calpain at 48 hours in the serum-containing culturesmade it impossible to purify intact, inactivated calpain at that timepoint (data not shown); accordingly, the use of the serum-free mediumfor the expression allowed a 3-4-fold increase in the startingconcentration of recombinant calpain for purification compared with thelevel at 24 hours in serum-containing cultures.

EXAMPLE 8 Enzymatic Activity of Independent 80 kDa Subunit

To determine the relative activity of the recombinantly produced 80kDasubunit, the enzymatic activity of the subunit was examined inunfractionated extracts from Sf21 cells infected with AcNPV-hCANPI80-5.1.5×10⁸ Sf21 cells were pelleted by centrifugation at 150×g, the mediumremoved, then resuspended with 3×10⁸ pfu of AcNPV-hCANPI80-5 virus in 10ml of supplemented Grace's medium with fetal bovine serum and incubatedfor 1 hr at 27° C. Following the incubation, the cells plus medium plusvirus were added to 90 ml of the same medium and incubated at 27° C. ina 250 ml spinner flask for 24 hours. Cells were harvested as in Example5 and the extract was examined for enzymatic activity also as describedin Example 5. The results from 33 μg of unfractionated extract aredepicted in FIG. 8. In FIG. 8, open squares represent activity withoutcalcium present. Open diamonds represent activity with calcium present.Open circles represent activity with calcium and calpain inhibitor Ipresent. The same amount of enzyme activity for both the recombinant 80kDa subunit and the recombinant calpain was then run on SDS-PAGE and theamount of each was qualitatively determined by immunoblot analysis asdescribed in Example 4 above. As determined therefrom, approximately 4-to 5-fold more isolated 80 kDa protein is needed to give activityequivalent to that of the heterodimeric protein. Thus, the specificactivity of the 80 kDa recombinant calpain I was experimentallydetermined to be approximately 20-25% that of the heterodimeric calpainI. This is approximately seven times greater than that of the 80 kDasubunit dissociated from native calpain I (Kikuchi et al., supra). Theactivity was also shown to be completely inhibited by 12.5 μM calpaininhibitor I, as is the heterodimer enzyme (FIG. 8).

EXAMPLE 9 Purification of Recombinant Enzymatically Active Calpain

As disclosed below, recombinant calpain was purified in four steps,including three chromatographic steps, to 94% purity as determined byreversed-phase HPLC analysis. Cell culture conditions were as describedin Example 7. The cells were lysed in a solution containing 10 mM HEPES,2 mM EDTA, 2 mM EGTA, 5 mM β-mercaptoethanol, 5 mM pepstatin, 0.1 mMPMSF, and 10 mg/ml aprotinin, pH 7.5 and homogenized using a 40 mlDounce homogenizer (Wheaton, Millville, N.J.). The material was thencentrifuged at 2,100×g for 10 minutes to pellet nuclei, followed bycentrifugation at 38,700×g for 1 hour to pellet membranes. Thesupernatant was precipitated with ammonium sulfate and proteins thatprecipitated between 30 to 451% ammonium sulfate were resuspended in abuffer solution containing 10 mM HEPES, 2 mM EDTA, 2 mM EGTA, 10 mMNaCl, and 5 mM β-mercaptoethanol, pH 7.5, dialyzed overnight against thesame buffer, and then separated on the following resins using standardtechniques: Q-Sepharose Fast Flow, followed by Phenyl Sepharose CL-4B(both from Pharmacia, Piscataway, N.J.), then Mimetic Red 2 (AmericanInternational Chemical, Natick, Mass.). Following this technique, 5-6 mgof highly-purified protein was isolated from 1 liter of cells. A15.5-fold purification was effected in three (3) chromatographicseparations to yield a protein with a high degree of purity.Purification of calpain from human erythrocytes required over a22,000-fold purification with four (4) chromatographic steps (Hatanakaet al., supra). This represents a major advantage of this recombinantexpression in being able to easily purify larger quantities of calpainthan can be easily done from native sources. For each analysis, enzymeactivity was determined by monitoring the rate of hydrolysis in thepresence of Ca²⁺ of the synthetic fluorogenic substrateSucc-Leu-Tyr-methoxyl-β-naphthylamine (Succ-Leu-Tyr-MNA) similarly tothe method used by Sasaki, T. et al., supra, for measuring hydrolysis ofSucc-Leu-Tyr-AMC. The experiments were performed in 96 well plates(Dynatech cat# 011-010=7905, 14340 Sullyfield Circle, Chantilly, Va.22021) and the fluorescence was detected using a 96 well plate readingfluorimeter (excitation=340 nM, emission=430 nM; Titertek Fluoroskan IIFinland).

EXAMPLE 10 Comparative Sensitivities of Native and RecombinantEnzymatically Active Calpain to Inhibitors

Native and recombinant enzymatically active calpain I were compared fortheir sensitivities to a number of known calpain I inhibitors. Toevaluate inhibitor sensitivities, stocks (40 times concentrated) of eachinhibitor to be tested were prepared in 100% anhydrous DMSO and 5 μl ofeach inhibitor preparation were aliquoted into each of three wells of a96 well plate. Dilutions of each enzyme preparation were made into assaybuffer (i.e., 50 mM Tris, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 5 mMβ-mercaptoethanol, pH 7.5 including 0.2 mM Succ-Leu-Tyr-MNA) and 175 μlof each dilution aliquoted into the same wells containing theindependent inhibitor stocks as well as to positive control wellscontaining 5 μl DMSO, but no inhibitor. To start the reaction, 20 μl of50 mM CaCl₂ in assay buffer was added to all wells of the plate,excepting three, which were used as background signal baseline controls.Substrate hydrolysis was monitored every 5 minutes for a total of 30minutes. Substrate hydrolysis in the absence of inhibitor was linear forup to 15 minutes. The rate of hydrolysis was determined as the change influorescence units per the 10 minute time period between 5 and 15minutes. At each inhibitor concentration tested, the percent inhibitionwas determined as the percent decrease in the rate of substratehydrolysis in the presence of inhibitor versus the rate in its absence.The 50% inhibition concentration (IC₅₀) determinations for threestructurally diverse known inhibitors of calpain--Z-Leu-Phe-CONHEt,Z-Leu-Leu-Phe-CH₂ S(+)Me₂ Br(-) and Z-Leu-Nle-H--are depicted in TableIII below. Note that the IC₅₀ s obtained for each calpain inhibitoragainst recombinant human calpain approximated those found for thenative enzyme. The rank order of inhibitor potency was the same.Prototypic inhibitors of serine (PMSF) and aspartic proteases (pepstatinA) were also included in this determination. Both the recombinant andnative enzymes showed insignificant inhibition of their activities bythese class specific inhibitors as exemplified by the 5-6 fold order ofmagnitude greater differences in the IC₅₀ values obtained with respectto the known calpain inhibitors.

                  TABLE III    ______________________________________    Inhibitor Profile                       Native  Recombinant                       (IC.sub.50, nM)    ______________________________________    Z--Leu--Phe--CONHEt  56        34    Z--Leu--Leu--Phe--CH.sub.2 S(+)Me.sub.2 Br(-)                         14         8    Z--Leu--Nle--H       14        10    Pepstatin A           >10,000   >10,000    PMSF                 >1,000,000                                   >1,000,000    ______________________________________

EXAMPLE 11 Comparison of Calcium Activation of Native and RecombinantCalpain

To determine the calcium concentration required for enzyme activity,tests were performed essentially as described by Kitahara et al., J.Biochem., 95:1759-1766 (1984). First, enzyme preparations were dialyzedovernight against 110 mM imidazole-HCl/1 mM EGTA buffer at pH 7.3containing 5 mM β-mercaptoethanol. Ten-fold concentrated Ca²⁺ /EGTAbuffers were prepared by adding varying amounts of CaCl₂ to theimidazole/EGTA buffer. Twenty μl of each buffer was put into three wellsof a 96 well plate. Dilutions of dialyzed enzyme were made into theimidazole/EGTA buffer containing 1 mM Succ-Leu-Tyr-MNA and 180 μl ofeach preparation were added to the wells containing the various Ca/EGTAbuffers. Substrate hydrolysis was measured every 5 minutes for 30minutes. The 1/2 V_(max) was determined as the rate of substratehydrolysis which was 50% of the maximal rate achieved in the presence ofthe varying amounts of calcium. The results are shown in FIG. 9. The 1/2V_(max) listed is an approximation of the Ca²⁺ ! based on the K_(d) ofEGTA for calcium in this buffer at the particular ionic strength, pH,and temperature (K_(d) =5.5×10⁻⁶ M). The concentration of calciumrequired to give 1/2 V_(max) was essentially the same for both nativecalpain and the recombinant calpain of this invention--i.e. 15 μM and 14μM, respectively. The Ca²⁺ ! activation profiles for both the native andrecombinant enzymes are virtually identical. In FIG. 9, open squareswith interior open circles represent recombinant human calpain I(rhCANPI). Shaded squares with interior open circles represent nativehuman calpain I (nhCANPI).

EXAMPLE 12 Assay for Calpain Inhibitors

Recombinant enzymatically active calpain is purified, for example, asdescribed in Example 9 above. The purified calpain can then be utilizedin an assay for screening potential inhibitors of calpain. The assayconditions can be similar to those described in Examples 9 and 10 above.For example, Succ-Leu-Tyr-MNA can be used as the substrate. Calpaininhibitor I can be used as a control for assaying inhibition of calpain.However, other substrates and known inhibitors can be utilized. (SeeSasaki, supra.) Samples without calpain inhibitor I present can be usedas enzyme activity controls. Each compound to be tested as a calpaininhibitor is assayed, for example, by the method described in Example 10where known inhibitors were assayed. However, other methods can beutilized.

The disclosures of all of the patents and publications discussed ordescribed herein are hereby incorporated by reference herein, in theirentirety.

The foregoing examples are meant to illustrate the invention and not tolimit it in any way. Those skilled in the art will recognize thatchanges can be made which are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 12    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CGGGATCCTTAGGAATACATAGTCAGCTGCAGCC34    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    CACCCTGATCTGAAGAC17    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GTACACTTGAAGCGTGACTTC21    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    CAGGCAGCAAACGAAATTGTC21    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CGGGATCCTTATGCAAACATGGTCAGCTGCAACC34    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    ATTTGCGGATGGTCCGGCTCTTGA24    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    CGCGGATCCTATAAATATGTCGGAGGAGATCATCACGCCG40    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    CCGGGATCCTATAAATATGTTCCTGGTT28    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    AACCAGGAACATATTTATAGGATC24    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GGTGGAACGGCCATGCGCATC21    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    CATTGATGATGGAGTCAGGAG21    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    CTGAGAACAGAGCCAAGAGA20    __________________________________________________________________________

We claim:
 1. An insect cell infected with a recombinant baculoviruscomprising cDNA encoding mammalian calpain I.
 2. The insect cell ofclaim 1 wherein said calpain is human calpain.
 3. The insect cellaccording to claim 1 wherein said baculovirus is Autographa californica.4. The insect cell according to claim 1 wherein said cell is of thespecies Spodoptera frugiperda.
 5. An insect cell infected with arecombinant baculovirus comprising cDNA encoding a subunit of mammaliancalpain I of about 80 kDa.
 6. The insect cell of claim 5 wherein saidcalpain is human calpain.
 7. A recombinant baculovirus having the ATCCdesignation ATCC VR
 2457. 8. A recombinant baculovirus having the ATCCdesignation ATCC VR
 2458. 9. An insect cell of the species Spodopterafrugiperda infected with a recombinant baculovirus of the speciesAutographa californica, said baculovirus comprising cDNA encodingmammalian calpain I.